Seed-Preferred Promoters

ABSTRACT

The present invention provides compositions and methods for regulating expression of nucleotide sequences in a plant. Compositions are novel nucleotide sequences for a tissue preferred promoter isolated from the  sorghum  α-kafirin coding region or from the  sorghum  β-kafirin coding region. The sequences drive expression preferentially to endosperm tissue. A method for expressing a nucleotide sequence in a plant using the regulatory sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a nucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed plant from the transformed plant cell.

CROSS-REFERENCE PARAGRAPH

This application claims the benefit of U.S. Provisional Application No.60/955,889, filed Aug. 15, 2007, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Expression of DNA sequences in a plant host is dependent upon thepresence of operably linked regulatory elements that are functionalwithin the plant host. Choice of the regulatory element will determinewhen and where within the organism the DNA sequence is expressed. Wherecontinuous expression is desired throughout the cells of a plant, and/orthroughout development, constitutive promoters are utilized. Incontrast, where gene expression in response to a stimulus is desired,inducible promoters are the regulatory element of choice. Whereexpression in specific tissues or organs are desired, tissue-preferredpromoters may be used. That is, they may drive expression in particulartissues or organs. Such tissue-preferred promoters may be temporallyconstitutive or inducible. With any of these variables, additionalregulatory sequences upstream and/or downstream from a core promotersequence may be included in expression constructs of transformationvectors to bring about varying levels of expression of nucleotidesequences in a transgenic plant.

Storage protein genes express in embryo and endosperm. In maize they arecalled zeins and at least about 41 members of the family have beenidentified in the maize genome. In sorghum the genes are referred to askafirin genes. Kafirins are classified by their structure, molecularweight and solubility characteristics. Thus α-kafirins have molecularweight of about 25,000 and 20,000, β-kafirins have molecular weight of20,000, 18,000 and 16,000 while γ-kafirins have a molecular weight of28,000. By way of example, sorghum α-kafirin genes have been identifiedby Reddy at GenBank accession number Y17555 (Eddy, E. N. P. “Sorghumbicolor var. INRA450 gene encoding alpha kafirin”) GenBank No. Y17555(1998)) and Y17556 (Reddy, E. N. P., “Sorghum bicolor var. White Martingene encoding alpha kafirin” (1998)). Kafirins have been of interestbecause, among other applications, they can influence digestibility ofsorghum grain. Thus there has been interest in modulating expression ofsuch genes.

As this field develops and more genes become accessible, a greater needexists for transformed plants with multiple genes. These multipleexogenous genes typically need to be controlled by separate regulatorysequences however. Further, some genes should be regulatedconstitutively whereas other genes should be expressed at certaindevelopmental stages or locations in the transgenic organism.Accordingly, a variety of regulatory sequences having diverse effects isneeded.

Diverse regulatory sequences are also needed as undesirable biochemicalinteractions can result from using the same regulatory sequence tocontrol more than one gene. For example, transformation with multiplecopies of a regulatory element may cause problems, such that expressionof one or more genes may be affected.

Isolation and characterization of a promoter that can serve as aregulatory element for expression of isolated nucleotide sequences ofinterest in a constitutive manner are needed for impacting varioustraits in plants. The inventors have isolated just such a promoter.

SUMMARY OF THE INVENTION

The invention is directed to a promoter from a Sorghum bicolorkafirin-encoding gene, specifically an α-kafirin-encoding and aβ-kafirin-encoding gene, both useful as a regulatory region andproviding for expression of an operably linked nucleotide sequence. Inan embodiment the expression is driven in an endosperm-preferred manner.The invention is further directed to functional fragments which functionis to drive endosperm preferred expression of an operably linkednucleotide sequences. Expression cassettes having the nucleotidesequence, plants expressing same, and methods of use in impactingexpression of operably linked nucleotides sequences are within the scopeof the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence of the sorghum α-kafirin promoter(SEQ ID NO: 1) with TATA box indicated.

FIG. 2 shows the nucleotide sequence of the sorghum β-kafirin promoter(SEQ ID NO: 2) with TATA box indicated.

DETAILED DESCRIPTION OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

The seed of a plant includes a seed coat, the embryo, and a supply ofstored food, the endosperm. The endosperm includes an aleurone layer,which refers to the granular protein found in the endosperm of manyseeds, and which forms the outermost layer in cereal grains. Cerealendosperm mature seed includes three cell types: the aleurone cells, thestarchy endosperm, and the basal endosperm transfer cells. The aleuroneis a layer of densely cytoplasmic cells covering the surface of theendosperm, just beneath the maternal pericarp tissue. When the seedgerminates, the aleurone cells are stimulated to secrete enzymes thatbreak down the storage compounds in the endosperm, allowing amino acidsand sugars to become available for update by the growing seedling, whichdevelops from the embryo.

In accordance with the invention, nucleotide sequences are provided thatallow regulation of transcription in an endosperm-preferred manner.Thus, the compositions of the present invention comprise novelnucleotide sequences for plant regulatory elements natively associatedwith the nucleotide sequences coding for Sorghum bicolor α-kafirinprotein, identified here as SB-AKAF. The compositions of the presentinvention also comprise novel nucleotide sequences for plant regulatoryelements natively associated with the nucleotide sequences coding forSorghum bicolor β-kafirin protein, identified here as SB-BKAF.

In an embodiment, the regulatory element drives transcription in anendosperm-preferred manner, wherein said regulatory element comprises anucleotide sequence selected from the group consisting of: a)sequencesnatively associated with, and that regulate expression of DNA coding forsorghum SB-AKAF (Sorghum bicolor α-kafirin protein); b) the nucleotidesequence set forth in SEQ ID NO: 1; c) sequences natively associatedwith, and that regulate expression of DNA coding for sorghum SB-BKAF(Sorghum bicolor β-kafirin protein) d) the nucleotide sequence set forthin SEQ ID NO: 2; or e) a sequence comprising a functional fragment ofthe nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

Further embodiments are to expression cassettes, transformation vectors,plants, and plant cells comprising the above nucleotide sequences. Theinvention is further to methods of using the sequence in plants andplant cells.

A method for modulating expression of an isolated nucleotide sequence ina plant using the regulatory sequences disclosed herein is provided. Themethod comprises transforming a plant cell with a transformation vectorthat comprises an isolated nucleotide sequence operably linked to one ormore of the plant regulatory sequences of the present invention andregenerating a stably transformed plant from the transformed plant cell.In this manner, the regulatory sequences are useful for controlling theexpression of endogenous as well as exogenous products in anendosperm-preferred manner.

At times it can be desirable to inhibit expression of a native DNAsequence within a plant's tissues to achieve a desired phenotype. Suchinhibition can be accomplished, by transformation of the plant with anexpression cassette comprising a tissue-preferred promoter operablylinked to an antisense nucleotide sequence, hairpin, RNA interfering orother nucleic acid molecule, such that tissue-preferred expression ofthe molecule interferes with translation of the mRNA of the native DNAsequence or otherwise inhibits expression of same in a subset of theplant's cells.

Under the regulation of the promoter will be a sequence of interest,which will provide for modification of the phenotype of the plant. Suchmodification includes, but is not limited to, modulating the productionof an endogenous product, as to amount, relative distribution, or thelike, or production of an exogenous expression product to provide for anovel function or product in the plant. Such a promoter is useful for avariety of applications, such as production of transgenic plants andseed with desired seed traits, including, but not limited to, alteredoil content, protein quality, cell growth or nutrient quality.

By “tissue-preferred” promoter or “endosperm-preferred” promoter isintended favored spatial expression in a specific tissue; here, favoredexpression in the endosperm of the seed.

By “regulatory element” is intended sequences responsible for expressionof the linked nucleic acid molecule including, but not limited topromoters, terminators, enhancers, introns, and the like.

By “promoter” is intended a regulatory region of DNA capable ofregulating the transcription of a sequence linked thereto. It usuallycomprises a TATA box capable of directing RNA polymerase II to initiateRNA synthesis at the appropriate transcription initiation site for aparticular coding sequence.

A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate and further include elements which impact spatial and temporalexpression of the linked nucleotide sequence. It is recognized thathaving identified the nucleotide sequences for the promoter regiondisclosed herein, it is within the state of the art to isolate andidentify further elements in the 5′ region upstream from the particularpromoter region. Thus, the promoter region disclosed here may compriseupstream elements such as those responsible for special and temporalexpression of the nucleic acid molecule, and may include: enhancers, theDNA response element for a transcriptional regulatory protein, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, activator sequences, and the like.

The promoter elements which enable expression can be identified,isolated, and used with other core promoter elements to conferendosperm-preferred expression. By core promoter elements is meant theminimal sequence required to initiate transcription, such as the TATAbox, which is common to promoters in genes encoding proteins. Thus theupstream promoter elements of SB-AKAF or SB-BKAF can optionally be usedin conjunction with its own or core promoter elements from othersources. The promoter may be native or non-native to the cell in whichit is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of a nucleotide sequence ofinterest. Less than the entire promoter region can be utilized and theability to drive tissue preferred expression retained. It is recognizedthat expression levels of mRNA can be modulated with specific deletionsof portions of the promoter sequence. Thus, the promoter can be modifiedto be a weak or strong promoter. Generally, by “weak promoter” isintended a promoter that drives expression of a coding sequence at a lowlevel. By “low level” is intended levels of about 1/10,000 transcriptsto about 1/100,000 transcripts to about 1/500,000 transcripts.Conversely, a strong promoter drives expression of a coding sequence ata high level, or at about 1/10 transcripts to about 1/100 transcripts toabout 1/1,000 transcripts. Generally, at least about 20 nucleotides ofan isolated promoter sequence will be used to drive expression of anucleotide sequence.

It is recognized that to increase transcription levels, enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′ UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon.

The term “isolated” refers to material, such as a nucleic acid orprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment, or (2) if the material is in itsnatural environment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art. One method is the use of primers andgenomic DNA used in conjunction with the Genome Walker Kit™ (Clonetech).

The SB-AKAF promoter set forth in SEQ ID NO: 1 is 858 nucleotides inlength. It expresses preferentially in endosperm from about 15 DAP (daysafter pollination) to seed maturity. The SB-AKAF promoter was identifiedby using the Sorghum bicolor α-kafirin coding region to assemble contigswhich contained flanking sequence, from the publicly available GenomeSurvey Sequence (GSS) collection. From the assembled in silico contig,PCR primers were designed which enabled isolation from sorghum genomicDNA.

The SB-BKAF promoter set forth in SEQ ID NO: 2 is 827 nucleotides inlength. It expresses preferentially in endosperm from about 12 DAP toseed maturity. The SB-BKAF promoter was isolated using the aboveprocedures used to identify and isolate the promoter from the Sorghumbicolor β-kafirin coding region.

The transcription regulating nucleotide sequences of the invention ortheir functional equivalents can be obtained or isolated from any plantor non-plant source, or produced synthetically by purely chemical means.

The regulatory regions of the invention may be isolated from any plant,including, but not limited to sorghum (Sorghum bicolor, Sorghumvulgare), corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.),alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean(Glycine max), tobacco (Nicotiana tabacum), millet (Panicum spp.),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), oats (Avena sativa), barley (Hordeum vulgare),vegetables, ornamentals, and conifers. Preferably, plants include corn,soybean, sunflower, safflower, canola, wheat, barley, rye, alfalfa, andsorghum.

Promoters isolated from one plant species can sometimes be expected toexpress in another plant species. For example, maize promoters have beenused to drive expression of genes in non-maize plants, including tobacco(Yang et al. (1990) “Maize sucrose synthase-1 promoter drives phloemcell-specific expression of GUS gene in transgenic tobacco plants” Proc.Natl. Acad. Sci. USA 87, 4144-4148; Geffers et al. (2000)“Anaerobiosis-specific interaction of tobacco nuclear factors withcis-regulatory sequences in the maize GapC4 promoter” Plant Mol. Biol.43:11-21); cultured rice cells (Vilardell et al., (1991) “Regulation ofthe maize rab 17 gene promoter in transgenic heterologous systems” PlantMol. Biol. 17:985-993), wheat (Oldach et al. (2001) “Heterologousexpression of genes mediating enhanced fungal resistance in transgenicwheat” Mol. Plant Microbe Interact. 14:832-838; Brinch-Pedersen et al.(2003) “Concerted action of endogenous and heterologous phytase onphytic acid degradation in seed of transgenic wheat (Triticum aestivumL.)” Transgenic Res. 12:649-659), rice (Cornejo et al. (1993) “Activityof a maize ubiquitin promoter in transgenic rice” Plant Mol. Biol.23:567-581; Takimoto et al. (1994) “Non-systemic expression of astress-response maize polyubiquitin gene (Ubi-1) in transgenic riceplants” Plant Mol. Biol. 26:1007-1012), sunflower (Roussell et al.(1988) “Deletion of DNA sequences flanking an Mr 19,000 zein genereduces its transcriptional activity in heterologous plant tissues” Mol.Gen. Genet. 211:202-209), and protoplasts of carrot (Roussell et al.,1988).

Regulatory sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the codingregion of the sequences set forth herein. In these techniques, all orpart of the known coding sequence is used as a probe which selectivelyhybridizes to other sequences present in a population of cloned genomicDNA fragments (i.e. genomic libraries) from a chosen organism. Methodsare readily available in the art for the hybridization of nucleic acidsequences. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions.Functional variants of the invention may be created by site-directedmutagenesis, induced mutation, or may occur as allelic variants(polymorphisms).

As used herein, a “functional fragment” is a regulatory sequence variantformed by one or more deletions from a larger regulatory element. Forexample, the 5′ portion of a promoter up to the TATA box near thetranscription start site can be deleted without abolishing promoteractivity, as described by Opsahl-Sorteberg et al., “Identification of a49-bp fragment of the HvLTP2 promoter directing aleruone cell specificexpression” Gene 341:49-58 (2004). Such fragments should retain promoteractivity, particularly the ability to drive expression in the preferredtissue. Activity can be measured by Northern blot analysis, or reporteractivity measurements when using transcriptional fusions, and the like.See, for example, Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′, 4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al. (1990) PCR Protocols, A Guideto Methods and Applications, eds., Academic Press). Primers used inisolating the promoter of the present invention are shown below.

The regulatory elements disclosed in the present invention, as well asvariants and fragments thereof, are useful in the genetic manipulationof any plant when operably linked with an isolated nucleotide sequenceof interest whose expression is to be controlled to achieve a desiredphenotypic response.

By “operably linked” is intended a functional linkage between aregulatory region and a second sequence, wherein the regulatory sequenceinitiates and mediates transcription of the DNA sequence correspondingto the second sequence.

The regulatory elements of the invention can be operably linked to theisolated nucleotide sequence of interest in any of several ways known toone of skill in the art. The isolated nucleotide sequence of interestcan be inserted into a site within the genome which is 3′ to thepromoter of the invention using site specific integration as describedin U.S. Pat No. 6,187,994. The term “nucleotide sequence of interest”refers to a nucleic acid molecule (which may also be referred to as apolynucleotide) which can be an RNA molecule as well as DNA molecule,and can be a molecule that encodes for a desired polypeptide or protein,but also may refer to nucleic acid molecules that do not constitute anentire gene, and which do not necessarily encode a polypeptide orprotein. For example, when used in a homologous recombination process,the promoter may be placed in a construct with a sequence that targetsan area of the chromosome in the plant but may not encode a protein. Ifdesired, the nucleotide sequence of interest can be optimized for planttranslation by optimizing the codons used for plants and the sequencearound the translational start site for plants. Sequences resulting inpotential mRNA instability can also be avoided.

The regulatory elements of the invention can be operably linked inexpression cassettes along with isolated nucleotide sequences ofinterest for expression in the desired plant. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the nucleotide sequence of interest under the transcriptional controlof the regulatory elements. Alternatively, a specific result can beachieved by providing for a reduction of expression of one or moreendogenous products, particularly enzymes or cofactors in the plant.This down regulation can be achieved through many different approachesknown to one skilled in the art, including antisense, co-suppression,use of hairpin formations, or others, and discussed infra. Importationor exportation of a cofactor also allows for control of plantcomposition. It is recognized that the regulatory elements may be usedwith their native or other coding sequences to increase or decreaseexpression of an operably linked sequence in the transformed plant orseed.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, and grain characteristics. Still other categories oftransgenes include genes for inducing expression of exogenous productssuch as enzymes, cofactors, and hormones from plants and othereukaryotes as well as prokaryotic organisms.

Modifications that affect grain traits include increasing the content ofoleic acid, or altering levels of saturated and unsaturated fatty acids.Likewise, the level of plant proteins, particularly modified proteinsthat improve the nutrient value of the plant, can be increased. This isachieved by the expression of such proteins having enhanced amino acidcontent.

Increasing the levels of lysine and sulfur-containing amino acids may bedesired as well as the modification of starch type and content in theseed. Hordothionin protein modifications are described in WO 94/16078filed Apr. 10, 1997; WO 96/38562 filed Mar. 26, 1997; WO 96/38563 filedMar. 26, 1997 and U.S. Pat. No. 5,703,409 issued Dec. 30, 1997. Anotherexample is lysine and/or sulfur-rich plant protein encoded by thesoybean 2S albumin described in WO 97/35023 filed Mar. 20, 1996, and thechymotrypsin inhibitor from barley, Williamson et al. (1987) Eur. J.Biochem. 165:99-106.

Agronomic traits in plants can be improved by altering expression ofgenes that: affect the response of plant growth and development duringenvironmental stress, Cheikh-N et al. (1994) Plant Physiol.106(1):45-51) and genes controlling carbohydrate metabolism to reducekernel abortion in maize, Zinselmeier et al. (1995) Plant Physiol.107(2):385-391.

It is recognized that any nucleotide sequence of interest, including thenative coding sequence, can be operably linked to the regulatoryelements of the invention and its expression modified in the plant.

Commercial traits in plants can be created through the modifiedexpression of genes that alter starch or protein for the production ofpaper, textiles, ethanol, polymers or other materials with industrialuses.

Means for increasing or inhibiting a protein are well known to thoseskilled in the art and, include, but are not limited to: transgenicexpression, antisense suppression, co-suppression, RNA interference,gene activation or suppression using transcription factors and/orrepressors; mutagenesis including, but not limited to, transposontagging; directed and site-specific mutagenesis, chromosome engineering(see Nobrega et. al., Nature 431:988-993(04)), homologous recombination,TILLING (Targeting Induced Local Lesions In Genomes), and biosyntheticcompetition to manipulate the expression of proteins. Many techniquesfor gene silencing are well known to one of skill in the art, includingbut not limited to knock-outs (such as by insertion of a transposableelement such as Mu, Vicki Chandler, The Maize Handbook ch. 118(Springer-Verlag 1994) or other genetic elements such as a FRT, Lox orother site specific integration site; RNA interference (Napoli et al.(1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323, Sharp (1999) GenesDev. 13:139-141, Zamore et al. (2000) Cell 101:25-33; and Montgomery etal. (1998) PNAS USA 95:15502-15507); virus-induced gene silencing(Burton, et al. (2000) Plant Cell 12:691-705, and Baulcombe (1999) Curr.Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff etal. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000)Nature 407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman &Sakai (2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al.(1992) EMBO J. 11:1525, and Perriman et al. (1993) Antisense Res. Dev.3:253); oligonucleotide mediated targeted modification (e.g., WO03/076574 and WO 99/25853); zinc-finger targeted molecules (e.g., WO01/52620; WO 03/048345; and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. (See,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065, 5,453,566, and 5,759,829). By “antisense DNA nucleotidesequence” is intended a sequence that is in inverse orientation to the5′-to-3′ normal orientation of that nucleotide sequence. When deliveredinto a plant cell, expression of the antisense DNA sequence preventsnormal expression of the DNA nucleotide sequence for the targeted gene.The antisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing with the endogenousmessenger RNA (mRNA) produced by transcription of the DNA nucleotidesequence for the targeted gene. In this case, production of the nativeprotein encoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the regulatory sequences disclosed herein canbe operably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant.

As noted, other potential approaches to impact expression of proteins inthe plant include traditional co-suppression, that is, inhibition ofexpression of an endogenous gene through the expression of an identicalstructural gene or gene fragment introduced through transformation(Goring, D. R., Thomson, L., Rothstein, S. J. 1991. Proc. Natl. Acad.Sci. USA 88:1770-1774 co-suppression; Taylor (1997) Plant Cell 9:1245;Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS USA91:3490-3496; Finnegan et al. (1994) Bio/Technology 12:883-888; andNeuhuber et al. (1994) Mol. Gen. Genet. 244:230-241)). In one example,co-suppression can be achieved by linking the promoter to a DNA segmentsuch that transcripts of the segment are produced in the senseorientation and where the transcripts have at least 65% sequenceidentity to transcripts of the endogenous gene of interest, therebysuppressing expression of the endogenous gene in said plant cell. (See,U.S. Pat. No. 5,283,184). The endogenous gene targeted forco-suppression may be a gene encoding any protein that accumulates inthe plant species of interest. For example, where the endogenous genetargeted for co-suppression is the 50 kD gamma-zein gene, co-suppressionis achieved using an expression cassette comprising the 50 kD gamma-zeingene sequence, or variant or fragment thereof.

Additional methods of down-regulation are known in the art and can besimilarly applied to the instant invention. These methods involve thesilencing of a targeted gene by spliced hairpin RNA's and similarmethods also called RNA interference and promoter silencing (see Smithet al. (2000) Nature 407:319-320, Waterhouse and Helliwell (2003)) Nat.Rev. Genet. 4:29-38; Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA95:13959-13964; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA97:4985-4990; Stoutjesdijk et al. (2002) Plant Phystiol. 129:1723-1731;and Patent Application WO 99/53050; WO 99/49029; WO 99/61631; WO00/49035 and U.S. Pat. No. 6,506,559.

For mRNA interference, the expression cassette is designed to express anRNA molecule that is modeled on an endogenous miRNA gene. The miRNA geneencodes an RNA that forms a hairpin structure containing a 22-nucleotidesequence that is complementary to another endogenous gene (targetsequence). miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants.

In one embodiment, the polynucleotide to be introduced into the plantcomprises an inhibitory sequence that encodes a zinc finger protein thatbinds to a gene encoding a protein of the invention resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a gene of the invention. Inother embodiments, the zinc finger protein binds to a messenger RNAencoding a protein and prevents its translation. Methods of selectingsites for targeting by zinc finger proteins have been described, forexample, in U.S. Pat. No. 6,453,242, and methods for using zinc fingerproteins to inhibit the expression of genes in plants are described, forexample, in U.S. Patent Publication No. 2003/0037355.

The regulatory region of the invention may also be used in conjunctionwith another promoter. In one embodiment, the plant selection marker andthe gene of interest can be both functionally linked to the samepromoter. In another embodiment, the plant selection marker and the geneof interest can be functionally linked to different promoters. In yetother embodiments, the expression vector can contain two or more genesof interest that can be linked to the same promoter or differentpromoters. For example, the SB-AKAF and SB-BKAF promoters described herecan be used to drive the gene of interest and the selectable marker, ora different promoter used for one or the other. Although an additionalpromoter may be the endogenous promoter of a structural gene ofinterest, the promoter can also be an exogenous regulatory sequence.

Other promoter elements can be those that are constitutive or sufficientto render promoter-dependent gene expression controllable as beingcell-type preferred, tissue-specific or time or developmental stagepreferred, or being inducible by external signals or agents. Suchelements may be located in the 5′ or 3′ regions of the gene. Promoterelements employed to control expression of product proteins and theselection gene can be any plant-compatible promoters. These can be plantgene promoters, such as, for example, the ubiquitin promoter(Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensenet al. (1992) Plant Mol. Biol. 18:675-689); the promoter for the smallsubunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzziet al., 1984; Broglie et al., 1984); or promoters from thetumor-inducing plasmids from Agrobacterium tumefaciens, such as thenopaline synthase, octopine synthase and mannopine synthase promoters(Velten, J. and Schell, J. (1985) “Selection-expression plasmid vectorsfor use in genetic transformation of higher plants” Nucleic Acids Res.13, 6981-6998; Depicker et al., (1982) Mol. and Appl. Genet. 1:561-573;Shaw et al. (1984) Nucleic Acids Research vol. 12, No. 20 pp 7831-7846)that have plant activity; or viral promoters such as the cauliflowermosaic virus (CaMV) 19S and 35S promoters (Guilley et al. (1982)“Transcription of Cauliflower mosaic virus DNA: detection of promotersequences, and characterization of transcripts” Cell 30:763-773; Odellet al. (1985) “Identification of DNA sequences required for activity ofthe cauliflower mosaic virus 35S promoter” Nature 313:810-812, thefigwort mosaic virus FLt promoter (Maiti et al. (1997) “Promoter/leaderdeletion analysis and plant expression vectors with the figwort mosaicvirus (FMV) full length transcript (FLt) promoter containing single ordouble enhancer domains” Transgenic Res. 6:143-156) or the coat proteinpromoter of TMV (Grdzelishvili et al., 2000) “Mapping of the tobaccomosaic virus movement protein and coat protein subgenomic RNA promotersin vivo” Virology 275:177-192).

The expression cassette may also include at the 3′ terminus of theisolated nucleotide sequence of interest, a transcriptional andtranslational termination region functional in plants. The terminationregion can be native with the promoter nucleotide sequence of thepresent invention, can be native with the DNA sequence of interest, orcan be derived from another source. Thus, any convenient terminationregions can be used in conjunction with the promoter of the invention,and are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions (Depicker etal., supra) or the pinII terminator from potato (An et al. (1989)“Functional analysis of the 3′ control region of the potatowound-inducible proteinase inhibitor II gene” Plant Cell 1:115-122). Seealso: Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshiet al. (1987) Nucleic Acid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al. (1986); MDMV leader (Maize Dwarf Mosaic Virus), Virology154:9-20; human immunoglobulin heavy-chain binding protein (BiP),Macejak et al. (1991) Nature 353:90-94; untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV), Gallie etal. (1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV), Lommel et al. (1991) Virology 81:382-385.See also Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have an expressed product ofan isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions such astransitions and transversions, can be involved.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample: Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al.(1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J.9:2517-2522; Kain et al. (1995) Bio Techniques 19:650-655; and Chiu etal. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to:genes encoding resistance to chloramphenicol, Herrera Estrella et al.(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella et al. (1983)Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian etal. (1995) Plant Science 108:219-227; streptomycin, Jones et al. (1987)Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard et al.(1996) Transgenic Res. 5:131-137; bleomycin, Hille et al. (1990) PlantMol. Biol. 7:171-176; sulfonamide, Guerineau et al. (1990) Plant Mol.Biol. 15:127-136; bromoxynil, Stalker et al. (1988) Science 242:419-423;glyphosate, Shaw et al. (1986) Science 233:478-481; phosphinothricin,DeBlock et al. (1987) EMBO J. 6:2513-2518.

Further, when linking a promoter of the invention with a nucleotidesequence encoding a detectable protein, expression of a linked sequencecan be tracked in the plant, thereby providing useful screenable orscorable markers. The expression of the linked protein can be detectedwithout the necessity of destroying tissue. By way of example withoutlimitation, the promoter can be linked with detectable markers includinga β-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (Jefferson et al., 1986, Proc.Natl. Acad. Sci. USA 83:8447-8451); chloramphenicol acetyl transferase;alkaline phosphatase; a R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., in Chromosome Structure and Function, KluwerAcademic Publishers, Appels and Gustafson eds., pp. 263-282 (1988);Ludwig et al. (1990) Science 247:449); a p-lactamase gene (Sutcliffe,Proc. Nat'l. Acad. Sci. U.S.A. 75:3737 (1978)), which encodes an enzymefor which various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l.Acad. Sci. U.S.A. 80:1101 (1983)), which encodes a catechol dioxygenasethat can convert chromogenic catechols; an α-amylase gene (Ikuta et al.,Biotech. 8:241 (1990)); a tyrosinase gene (Katz et al., J. Gen.Microbiol. 129:2703 (1983)), which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone, which in turn condenses toform the easily detectable compound melanin a green fluorescent protein(GFP) gene (Sheen et al., Plant J. 8(5):777-84 (1995)); a lux gene,which encodes a luciferase, the presence of which may be detected using,for example, X-ray film, scintillation counting, fluorescentspectrophotometry, low-light video cameras, photon counting cameras ormultiwell luminometry (Teeri et al. (1989) EMBO J. 8:343); DS-REDEXPRESS (Matz et al. (1999) Nature Biotech. 17:969-973, Bevis et al.(2002) Nature Biotech 20:83-87, Haas et al. (1996) Curr. Biol.6:315-324); Zoanthus sp. yellow fluorescent protein (ZsYellow) that hasbeen engineered for brighter fluorescence (Matz et al. (1999) NatureBiotech. 17:969-973, available from BD Biosciences Clontech, Palo Alto,Calif., USA, catalog no. 632443); and cyan florescent protein (CYP)(Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002)Plant Physiol 129:913-42).

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleicacids). Vectors and procedures for cloning and expression in E. coli arediscussed in Sambrook et al. (supra).

The transformation vector comprising the regulatory sequence of thepresent invention operably linked to an isolated nucleotide sequence inan expression cassette, can also contain at least one additionalnucleotide sequence for a gene to be cotransformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector. Vectors that are functional in plants can bebinary plasmids derived from Agrobacterium. Such vectors are capable oftransforming plant cells. These vectors contain left and right bordersequences that are required for integration into the host (plant)chromosome. At minimum, between these border sequences is the gene to beexpressed under control of the regulatory elements of the presentinvention. In one embodiment, a selectable marker and a reporter geneare also included.

A transformation vector comprising the particular regulatory sequencesof the present invention, operably linked to an isolated nucleotidesequence of interest in an expression cassette, can be used to transformany plant. In this manner, genetically modified plants, plant cells,plant tissue, and the like can be obtained.

Transformation protocols can vary depending on the type of plant orplant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of transforming plant cells include, but are notlimited to, microinjection, Crossway et al. (1986) Biotechniques4:320-334; electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci.USA 83:5602-5606; Agrobacterium-mediated transformation, see forexample, Townsend et al., U.S. Pat. No. 5,563,055; direct gene transfer,Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particleacceleration, see for example, Sanford et al., U.S. Pat. No. 4,945,050,Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);and McCabe et al. (1988) Biotechnology 6:923-926. Also see Weissinger etal. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Bio/Technology8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839; Hooydaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad.Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in TheExperimental Manipulation of Ovule Tissues, ed. G. P. Chapman et al.(Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou et al. (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens).

The cells that have been transformed can be grown into plants inaccordance with conventional methods. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants can then be grown andpollinated with the same transformed strain or different strains. Theresulting plant having constitutive expression of the desired phenotypiccharacteristic can then be identified. Two or more generations can begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Isolation of Regulatory Sequences

Regulatory regions from sorghum SB-AKAF (Sorghum bicolor α-kafirinprotein) and SB-BKAF (Sorghum bicolor β-kafirin protein) were isolatedfrom sorghum plants and cloned. Sorghum SB-AKAF or SB-BKAF was selectedas a source of endosperm-preferred regulatory elements based on thespatial and temporal expression of its products.

The SB-AKAF promoter is an 858 base pair nucleotide sequence (SEQ ID NO:1), shown in FIG. 1 with the TATA box underlined. A BLAST of GenBankshowed the highest identity was 100% to a subset of sequences within a90923 base pair Sorghum bicolor bacterial clone, GenBank accessionnumber: AF527808.1 (GI:22208471 by Song et al. “Sorghum bicolor cloneBAC SBTXS_(—)0040L6 php200725 orthologous region” (2002)); to a regionof a 75840 base pair BAC, GenBank accession number: AF527807.1(GI:22208458 by Song et al. “Sorghum bicolor clone BAC SB_BBc0126P21php200725 orthologous” (2002)), and to the two Sorghum bicolor genesencoding α-kafirin by Reddy, supra. None of these references identifiedthe promoter.

The SB-BKAF promoter is an 827 base pair nucleotide sequence (SEQ ID NO:2), shown in FIG. 2 with the TATA box underlined.

The method for isolation of the above sequences was as follows:

The SB-AKAF promoter was isolated from sorghum genomic DNA by using thesequences from the GSS database to design primers for PCR. The primersfor SB-AKAF PRO PCR were:

(SEQ ID NO 3) GGTTACCGAATTCTCAATAGCTATAGTTCAACAAATACTCCCTCC (SEQ ID NO4) CCATGGTTGTGAGGACGTTGACGTTTGTTTCCCTTGGTATG

The SB-BKAF promoter was isolated from sorghum genomic DNA by using thesequences from the GSS database to design primers for PCR. The primersfor SB-BKAF PRO PCR were:

(SEQ ID NO 5) CGGACCGGGTTACCGAATTCGGCCTGTTTGGTTTATGCCCAAATTTGCCA TAC(SEQ ID NO 6) GGTCTCTCATGCTACACCTGATCTGTTGAGCGCTCCTCTCTACGGCTCTGCTTTGCTTGC(Underlined portions indicate region of primer not homologous to thetemplate. This is used to add restriction enzyme sites to facilitatecloning)

The PCR reactions were performed in a Bio-Rad icycler (Hercules, Calif.)thermal cycler using Hifidelity supermix (Cat. # 10790-020, LifeTechnologies, Rockville Md.). The following cycle parameters were used:94° C. for 2 seconds, followed by 30 cycles of 94° C. for 20 seconds,55° C. for 30 seconds, and 68° C. for 1 minute. Finally, the sampleswere held at 67° C. for 4 minutes and then at 4° C. until furtheranalysis.

The PCR products were cloned into the (Promega™) pGEM-easy vector andsequenced using M13F and M13R primers. Upon sequence verification, theywere given PHP numbers and archived. Once amplified, the PCR fragmentswere sequenced and assembled into expression cassettes using the DS-REDEXPRESS coding region (supra) or YFP coding region (supra) as the markergene.

Example 2 Expression Data Using Promoter Sequences

A construct, named PHP29022 was prepared which included the sorghumα-kafirin promoter with the DS-RED EXPRESS selectable marker, supra andthe pinII terminator from potato (An et al., 1989 supra).

Another construct, PHP29281, was prepared which included the sorghumβ-kafirin promoter, along with the YELLOW FLUORESCENT PROEIN (YFP)marker (supra) and the nopaline synthase or nos promoter, supra. Allvectors were constructed using standard molecular biology techniques(Sambrook et al., supra). Successful subcloning was confirmed byrestriction analysis.

Example 3 Transformation and Regeneration of Maize Callus viaAgrobacterium

Constructs used were as those set forth supra using a binary plasmidwith the left and right borders (see Hiei et al., U.S. Pat. No.7,060,876) and the selectable marker for maize-optimized PAT(phosphinothricin acetyl transferase). Jayne et al., U.S. Pat. No.6,096,947.

Preparation of Agrobacterium Suspension:

Agrobacterium was streaked out from a −80° frozen aliquot onto a platecontaining PHI-L medium and was cultured at 28° C. in the dark for 3days. PHI-L media comprises 25 ml/l Stock Solution A, 25 ml/l StockSolution B, 450.9 ml/l Stock Solution C and spectinomycin (SigmaChemicals) was added to a concentration of 50 mg/l in sterile ddH2O(stock solution A: K2HPO4 60.0 g/l, NaH2PO4 20.0 g/l, adjust pH to 7.0w/KOH and autoclaved; stock solution B: NH4Cl 20.0 g/l, MgSO4.7H2O 6.0g/l, KCl 3.0 g/l, CaCl2 0.20 g/l, FeSO4.7H2O 50.0 mg/l, autoclaved;stock solution C: glucose 5.56 g/l, agar 16.67 g/l (#A-7049, SigmaChemicals, St. Louis, Mo.) and was autoclaved).

The plate can be stored at 4° C. and used usually for about 1 month. Asingle colony was picked from the master plate and was streaked onto aplate containing PHI-M medium [yeast extract (Difco) 5.0 g/l; peptone(Difco)10.0 g/l; NaCl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8, containing50 mg/L spectinomycin] and was incubated at 28° C. in the dark for 2days.

Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l,Eriksson's vitamin mix (1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l (Sigma); 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma) 1.5 mg/l;L-proline (Sigma) 0.69 g/l; sucrose (Mallinckrodt) 68.5 g/l; glucose(Mallinckrodt) 36.0 g/l; pH 5.2] for the PHI basic medium system, orPHI-I [MS salts (GIBCO BRL) 4.3 g/l; nicotinic acid (Sigma) 0.5 mg/l;pyridoxine.HCl (Sigma) 0.5 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol(Sigma) 0.10 g/l; vitamin assay casamino acids (Difco Lab) 1 g/l; 2,4-D1.5 mg/l; sucrose 68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2 w/KOHand filter-sterilize] for the PHI combined medium system and 5 ml of 100mM (3′-5′-Dimethoxy-4′-hydroxyacetophenone, Aldrich chemicals) was addedto a 14 ml Falcon tube in a hood. About 3 full loops (5 mm loop size)Agrobacterium was collected from the plate and suspended in the tube,then the tube vortexed to make an even suspension. One ml of thesuspension was transferred to a spectrophotometer tube and the OD of thesuspension is adjusted to 0.72 at 550 nm by adding either moreAgrobacterium or more of the same suspension medium, for anAgrobacterium concentration of approximately 0.5×109 cfu/ml to 1×109cfu/ml. The final Agrobacterium suspension was aliquoted into 2 mlmicrocentrifuge tubes, each containing 1 ml of the suspension. Thesuspensions were then used as soon as possible.

Embryo Isolation, Infection and Co-Cultivation:

About 2 ml of the same medium (here PHI-A or PHI-I) which is used forthe Agrobacterium suspension was added into a 2 ml microcentrifuge tube.Immature embryos were isolated from a sterilized ear with a sterilespatula (Baxter Scientific Products S1565) and dropped directly into themedium in the tube. A total of about 100 embryos are placed in the tube.The optimal size of the embryos was about 1.0-1.2 mm. The cap was thenclosed on the tube and the tube vortexed with a Vortex Mixer (BaxterScientific Products S8223-1) for 5 sec. at maximum speed. The medium wasremoved and 2 ml of fresh medium were added and the vortexing repeated.All of the medium was drawn off and 1 ml of Agrobacterium suspension wasadded to the embryos and the tube is vortexed for 30 sec. The tube wasallowed to stand for 5 min. in the hood. The suspension of Agrobacteriumand embryos was poured into a Petri plate containing either PHI-B medium[CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's vitamin mix(1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; silver nitrate 0.85 mg/l; gelrite (Sigma) 3.0 g/l;sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], for the PHI basicmedium system, or PHI-J medium [MS Salts 4.3 g/l; nicotinic acid 0.50mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol100.0 mg/l; 2,4-D 1.5 mg/l; sucrose 20.0 g/l; glucose 10.0 g/l;L-proline 0.70 g/l; MES (Sigma) 0.50 g/l; 8.0 g/l agar (Sigma A-7049,purified) and 100 mM acetosyringone with a final pH of 5.8 for the PHIcombined medium system. Any embryos left in the tube were transferred tothe plate using a sterile spatula. The Agrobacterium suspension wasdrawn off and the embryos placed axis side down on the media. The platewas sealed with Parafilm tape or Pylon Vegetative Combine Tape (productnamed “E.G.CUT” and is available in 18 mm×50 m sections; Kyowa Ltd.,Japan) and was incubated in the dark at 23-25° C. for about 3 days ofco-cultivation.

Resting, Selection and Regeneration Steps:

For the resting step, all of the embryos were transferred to a new platecontaining PHI-C medium [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l;Eriksson's vitamin mix (1000× Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer(Sigma) 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silver nitrate0.85 mg/l; carbenicillin 100 mg/l; pH 5.8]. The plate was sealed withParafilm or Pylon tape and incubated in the dark at 28° C. for 3-5 days.

Longer co-cultivation periods may compensate for the absence of aresting step since the resting step, like the co-cultivation step,provides a period of time for the embryo to be cultured in the absenceof a selective agent. Those of ordinary skill in the art can readilytest combinations of co-cultivation and resting times to optimize orimprove the transformation.

For selection, all of the embryos were then transferred from the PHI-Cmedium to new plates containing PHI-D medium, as a selection medium,[CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's vitamin mix(1000× Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer 0.5 g/l; agar (SigmaA-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN,Costa Mesa, Calif.) 100 mg/l; bialaphos (Meiji Seika K. K., Tokyo,Japan) 1.5 mg/l for the first two weeks followed by 3 mg/l for theremainder of the time.; pH 5.8] putting about 20 embryos onto eachplate.

The plates were sealed as described above and incubated in the dark at28° C. for the first two weeks of selection. The embryos weretransferred to fresh selection medium at two-week intervals. The tissuewas subcultured by transferring to fresh selection medium for a total ofabout 2 months. The herbicide-resistant calli are then “bulked up” bygrowing on the same medium for another two weeks until the diameter ofthe calli is about 1.5-2 cm.

For regeneration, the calli were then cultured on PHI-E medium [MS salts4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l, thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, Zeatin 0.5 mg/l,sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l, Indoleacetic acid (IAA,Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma) 0.1 mM, Bialaphos 3 mg/l,carbenicillin 100 mg/l adjusted to pH 5.6] in the dark at 28° C. for 1-3weeks to allow somatic embryos to mature. The calli were then culturedon PHI-F medium (MS salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5mg/l; sucrose 40.0 g/l; gelrite 1.5 g/l; pH 5.6] at 25° C. under adaylight schedule of 16 hrs. light (270 uE m-2sec-1) and 8 hrs. darkuntil shoots and roots are developed. Each small plantlet was thentransferred to a 25×150 mm tube containing PHI-F medium and is grownunder the same conditions for approximately another week. The plantswere transplanted to pots with soil mixture in a greenhouse. DS-REDEXPRESS events and YFP events were determined at regenerated plant stageby dissection of kernel tissue.

Ability of the SB-AKAF and SB-BKAF promoter to drive expression in maizewas confirmed by DS-RED EXPRESS or YFP detection in plant tissue by theprocedures outlined supra.

It was determined that the SB-AKAF PRO drove expression in maize inendosperm from about 15 DAP (days after pollination) to seed maturity.No leaf expression was observed.

The SB-BKAF PRO drove expression in maize endosperm from about 12 DAP toseed maturity. No leaf expression was observed.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. All referencescited are incorporate herein by reference.

1. An isolated promoter that drives transcription in a seedendosperm-preferred manner, wherein the promoter comprises a nucleotidesequence selected from the group consisting of: a) the nucleotidesequence of SEQ ID NO: 1 or 2; and b) a sequence comprising a functionalfragment of the nucleotide sequence set forth in a)
 2. An expressioncassette comprising a promoter operably linked to a nucleotide sequencewherein the promoter comprises the nucleotide sequence of claim
 1. 3. Aplant stably transformed with an expression cassette of claim
 2. 4. Theplant of claim 3, wherein said plant is a monocot.
 5. The plant of claim3, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.6. Seed of the plant of claim 3 wherein the seed comprises theexpression cassette.
 7. A method for selectively expressing a nucleotidesequence in a plant cell, the method comprising: a) transforming a plantcell with an expression cassette, the expression cassette comprising apromoter operably linked to a nucleotide sequence wherein the promotercomprises a nucleotide sequence selected from the group consisting of:i) the nucleotide sequence set forth in SEQ ID NO: 1 or 2; and ii) afunctional fragment of i); and b) growing the plant cell to selectivelyexpress the nucleotide sequence.
 8. The method of claim 7 wherein thepromoter initiates expression of the nucleotide sequence in seedendosperm tissue.
 9. The method of claim 7 further comprisingregenerating a stably transformed plant from the plant cell; whereinexpression of the nucleotide sequence alters the phenotype of a plant.10. The plant of claim 9, wherein said plant is a monocot.
 11. The plantof claim 9, wherein said monocot is maize, wheat, rice, barley, sorghum,or rye.
 12. Seed of the plant of claim 9 wherein the seed comprises theexpression cassette.